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The Journal of Immunology, 2003, 170: 2680-2687.
Copyright © 2003 by The American Association of Immunologists

Synthesis and Secretion of Monocyte Chemotactic Protein-1 Stimulated by the High Affinity Receptor for IgE

Santa Eglite, Juan M. Morin1 and Henry Metzger2

Section on Chemical Immunology, Arthritis and Rheumatism Branch, National Institute of Arthritis and Musculoskeletal Diseases, National Institutes of Health, Bethesda, MD 20892


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In prior studies aggregation of the high affinity receptors for IgE, Fc{epsilon}RI, on a rat mast cell line, RBL-2H3, stimulated transcription of the gene for monocyte chemotactic protein-1 (MCP-1) and secretion of the protein. Unexpectedly, those delayed events appeared much less constrained by kinetic proofreading than had been documented for other receptor-initiated responses. The results of the present experiments are consistent with the proposal that the biosynthesis and secretion of MCP-1 result from a soluble messenger formed in the reaction cascades initiated by the receptor, and that Ca2+ could serve as that messenger. Interestingly, whereas receptor-mediated signals were required for transcription of the gene for MCP-1 and secretion of the chemokine, such signals were not required for the intervening step of translation of its mRNA.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Many of the responses of cells of the rat basophilic leukemia line 2H3 (RBL-2H3) initiated by aggregation of their receptors with high affinity for IgE (Fc{epsilon}RI) are constrained by a kinetic proofreading (1) regimen. That is, delayed responses are disproportionately diminished compared with early ones when the cells are exposed to ligands that dissociate relatively rapidly from the receptor-bound IgE (2, 3). An apparent exception was observed with respect to the stimulated transcription of the gene for monocyte chemotactic protein-1 (MCP-1)3 (1). In this instance, doses of a rapidly dissociating, weakly binding Ag, which stimulated degranulation only poorly, stimulated the more delayed transcription of the MCP-1 gene almost as well as the homologous Ag with a much lower off-rate constant.

Our provisional interpretation of this phenomenon was that whereas certain biochemical pathways stimulated by Fc{epsilon}RI, such as those leading to degranulation and the phosphorylation of cellular constituents, such as Pyk2 and Erk2 (2), appear to involve multiple steps that required persistence of the particular liganded receptors that initiated the sequence of events, other pathways, such as those stimulating transcription of the gene for MCP-1 might not be so constrained. We suggested that a form of liganded receptor promotes the formation of a component (X') that, in turn, stimulates distal steps in a separate pathway without having to be physically associated with a liganded receptor (3). Because the concentration of this component is only maintained by the continued presence of receptor-associated intermediates, the system would nevertheless remain sensitive to the state of ligation of the population of receptors as a whole. An accompanying paper described a formal analysis of such a branch that escapes the kinetic proofreading that governs the main pathway (4).

We also indicated that Ca2+ was a plausible candidate for a component such as X', citing as evidence that Ca2+-ATPase inhibitors, which raise intracellular Ca2+, promote both transcription of the gene for MCP-1 and secretion of the chemokine (5, 6). In this paper we follow up on this proposal and explore how ligand affinity influences the changes in intracellular Ca2+ concentration ([Ca2+]i) induced by aggregation of Fc{epsilon}RI and the consequences with respect to the generation and secretion of MCP-1.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials

The anti-DNP monoclonal mouse IgE and its purification have been described previously (7, 8). The Ags were from a single batch of Fab of bovine IgG conjugated alternatively with DNP- or 2-nitrophenyl (NP)-NH2-caproate at a ratio of ~3 mol of hapten/mol of protein (3). Goat anti-mouse IgE was affinity-purified and rendered monospecific on appropriate columns of IgE. Mouse monoclonal IgG2b anti-phosphotyrosine (24G2) conjugated with biotin was purchased from Upstate Biotechnology (Lake Placid, NY), and HRP-extravidin was obtained from Sigma-Aldrich (St. Louis, MO). Fura Red acetoxymethyl ester and fluo-4 acetoxymethyl ester were purchased from Molecular Probes (Eugene, OR). Actinomycin D, U-73122, U-73343, bisindolylmaleimide V, Ro-31-8425, and Ro-31-8220 were purchased from Calbiochem (San Diego, CA). The hapten N-2,4-DNP-{epsilon}-amino-N-caproic acid and cyclopiazonic acid (CPA) were obtained from Sigma-Aldrich. 2,5-Di-tert-butylhydroquinone (DTBHQ) was purchased from Aldrich (Milwaukee, WI). Buffer A contained 150 mM NaCl, 5 mM KCl, 25 mM PIPES, 5.4 mM sucrose, 1 mM MgCl2, 1.8 mM CaCl2 (pH 7.2), and 0.1% BSA.

Cell culture and sensitization with IgE

RBL-2H3 cells were maintained as previously described (9). For experiments, cells were grown to confluence and used ~36 h after plating. Except for experiments involving measurements of Ca2+, cells were sensitized overnight by adding 0.5 µg/ml anti-DNP-specific mouse IgE to the medium. The next day cells were harvested using trypsin. For the studies involving measurements of Ca2+, the cells were suspended at 5 x 106 cells/ml in culture medium supplemented with 5 µg/ml DNP-specific IgE and incubated at room temperature for 1 h on a rotary mixer.

Detection of phosphorylated Fc{epsilon}RI

Cells were suspended in buffer A at a cell density of 1 x 106 cells/ml. After stimulation at 37°C with varying doses of DNP or NP Ags, the cells were lysed with Triton X-100-containing 3x solubilization buffer, anti-mouse IgE was added, and the immunoprecipitated Fc{epsilon}RI:IgE complexes were analyzed for phosphotyrosine as previously described (10).

Changes in [Ca2+]I

Confluent RBL-2H3 cells were harvested by treatment with trypsin. After sensitization (above) of the cells (5 x 106/ml), all cells to be used in a given experiment were loaded in one batch with Fura Red and fluo-4 by incubation with 9.2 µM acetoxymethyl esters of each dye (prepared as 2 mg/ml DMSO stock solutions) at room temperature for 45 min in the dark. The cells were washed twice with cold buffer A, resuspended in fresh buffer A at 0.67 x 106/ml, and kept on ice until warmed to room temperature or 37°C before measurements of changes in [Ca2+]i by flow cytometry. Control experiments showed that maintaining the cells for prolonged periods at room temperature led to a gradual leakage of the fluorescent indicators, but this could be prevented by keeping the cells on ice. Baseline measurements were followed by addition of the NP or DNP Ags, and the responses were monitored for 5 min. The collected data were processed using the FCSAssistant 2.1.6 {beta} program (http://www.fcspress.com) to determine the Fluo-4/Fura Red fluorescence intensity ratio, an indicator of intracellular Ca2+ (11). Control experiments showed that for cells kept on ice for up to 138 min there was no alteration in their baseline fluorescent ratio compared with cells examined immediately and only a small decrease in their response to stimulation with Ag. More detailed control experiments showed that the height of the initial peak of the Ca2+ response fell by an average of 2% in successive experiments, probably due to the ~12-min longer time that the cells were kept on ice for the successive doses of Ag. This factor should be considered when viewing the data on [Ca2+]i in response to varying concentrations of DNP and NP Ags. The experiments were always performed starting with the highest concentrations of DNP Ag and alternating the DNP and NP Ags in successive runs. Thus, with three doses the response to the lowest dose of NP would be underestimated by ~12%; with five doses, the response would be underestimated by 20%.

Production of MCP-1

Cells that had been sensitized overnight (described above) were harvested, resuspended at 5 x 106 cells/ml in culture medium, and incubated for 1 h at room temperature on a rotary mixer. Cells were stimulated at a density of 1 x 106 cells/ml in culture medium containing 2% FCS instead of the 17% present in the growth medium. In the experiments studying the MCP-1 response in the absence of external Ca2+, we used medium from which CaCl2 was omitted and substituted gelatin (0.05%) for FCS. (Control experiments showed that cells stimulated in the absence of FCS, but with added Ca2+, responded normally.) Cells were stimulated in a water bath at 37°C, flicking the tubes every 5–10 min to resuspend any sedimented cells. Total MCP-1 was measured in lysates prepared from aliquots of the cell suspension reacted with 0.03% Triton X-100. Secreted MCP-1 was measured in the supernatants of similarly sized aliquots of the suspensions that had simply been centrifuged. In the experiments in which cells were examined over 4–5 h, only ~85% of the initial cells in the suspension were recovered, probably due to progressive adherence of the remainder. In these later samples, the total MCP-1 produced would be underestimated by ~15%, whereas the amount secreted would not be equivalently underestimated. MCP-1 was measured using the rat MCP-1 ELISA kit supplied by BioSource (Camarillo, CA) according to the manufacturer’s protocols. To quantify MCP-1 in cell lysates, the standards were adjusted to contain the same concentration of Triton X-100 as was present in the samples. All data were derived within the linear range of the ELISA by diluting the samples as needed.

Most experiments were repeated at least three times to test for reproducibility. However, although we attempted to handle the cells in the same way, the absolute picograms of MCP-1 varied on a day-to-day basis. For this reason we present absolute data only from individual experiments and relative (normalized) data where the results of several experiments were pooled.

Hexosaminidase assay

The activity of hexosaminidase was measured as previously described (12), using p-nitrophenyl N-acetyl-{beta}-D-glucosaminide as a substrate.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Phosphorylation of tyrosines on Fc{epsilon}RI

The initial experiments were designed to determine the concentrations of high and low affinity Ags that stimulate quantitatively similar phosphorylation of Fc{epsilon}RI. Phosphotyrosines on the receptor were measured 3 min after the cells were exposed to an Ag, the approximate time that phosphorylation reached a maximum under the experimental conditions we used (not shown). Fig. 1 shows the results of experiments in which the IgE-sensitized cells were reacted with varying doses of DNP ({square}) or NP ({triangleup}) Ags. All data were normalized to the maximum phosphotyrosine observed with 20 ng/ml DNP conjugate in the individual experiments. For the lower doses, ~10–20 times higher concentrations of NP-Fab over DNP-Fab were needed to obtain comparable levels of phosphorylation of the receptor. The same relative concentrations of Ags were required in our prior study in which phosphorylation of Fc{epsilon}RI in response to those Ags was determined for adherent RBL-2H3 cells (3), although the maximal concentration of phosphorylated Fc{epsilon}RI was observed at 30, rather than 3, min. However, in the present study for the highest doses of both Ags tested (50 and 500 ng/ml, respectively), the DNP-bearing Ag stimulated somewhat higher phosphorylation of the receptor than the NP-bearing Ag.



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  		FIGURE 1. Dose-dependent phosphorylation of Fc{epsilon}RI. Cells sensitized with anti-DNP IgE were stimulated at 37°C for 3 min with varying doses of Ag. The Fc{epsilon}RI were then immunoprecipitated, and phosphorylated tyrosines were detected by Western blotting and quantified by densitometry of the films. The data have been normalized relative to the maximum signal obtained in samples stimulated with 20 ng/ml DNP conjugate. Upper abscissa, Dose of DNP Ag ({square}); lower abscissa, dose of NP Ag ({triangleup}). Mean values (±range) from two experiments are shown.

 
In these studies, which involved many more experiments than those shown in Fig. 1, we regularly observed that compared with the DNP Ag, the NP Ag stimulated a somewhat reduced ratio of phosphotyrosine on the dimer of {gamma}-chains relative to the {beta} subunit. Accurate quantitation of this effect requires careful correction for any influence on the measured ratios of the absolute intensity of the respective bands on the films. We defer to another report our documentation of this apparent difference (C. Torigoe et al., manuscript in preparation)

Changes in [Ca2+]i

At concentrations that stimulate detectable phosphorylation of the Fc{epsilon}RI, addition of the high affinity, slowly dissociating DNP Ag to RBL-2H3 cells primed with anti-DNP IgE stimulated an increase in free intracellular calcium (Fig. 2A). At higher doses there was an initial abrupt elevation followed by a more gradual decrease in [Ca2+]i with time; at lower doses the maximum increase was delayed, and [Ca2+]i remained augmented for a longer period. Qualitatively, the low affinity, rapidly dissociating NP Ag stimulated similar changes, although the dose-related differences were accentuated (Fig. 2B). The persistent elevation of [Ca2+]i at the lower, compared with the higher, doses is noteworthy. This phenomenon, i.e., that persistent elevation was more prominent at lower doses, clarifies why at doses of the two Ags that yielded roughly equivalent initial elevations of [Ca2+]i, the rise in [Ca2+]i persisted longer in response to the high affinity Ag (see, for example, the control curves in Fig. 3A).



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  		FIGURE 2. Intracellular Ca2+ in RBL-2H3 cells stimulated with Ag. Cells sensitized with anti-DNP IgE were stimulated at 37°C with varying doses of high and low affinity Ags (A, 50, 10, or 2.5 ng/ml DNP Ag; B, 500, 100, or 25 ng/ml NP Ag). In all experiments the Ag was added at 100 s, and the changes in [Ca2+]i were recorded for the following 5 min. One representative experiment of three is shown.

 


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  		FIGURE 3. Effects of different additives on Ag-induced Ca2+ response. A, Five nanograms of DNP or 200 ng NP Ag was added at 100 s to all samples, and 2 mM EGTA was added to some. B, Cells were exposed, or not, to 100 µM LaCl3 immediately before being stimulated with 50 ng/ml DNP Ag at 100 s. The baseline values fell somewhat when LaCl3 was added to the cells. For this reason both baselines are shown in this panel. C, Cells were stimulated with 50 ng/ml DNP Ag. At the peak of the Ca2+ response, 100 µM hapten was added to some of the samples. All experiments were conducted at room temperature. The Ca2+ response was monitored for a total of 5 min following stimulation with Ag. The results are from one of two experiments with very similar findings.

 
To study the role of calcium signaling in the biosynthesis MCP-1 further, we inhibited calcium influx into the cells by limiting extracellular Ca2+, depleting it with EGTA, blocking its entry with LaCl3, or aborting receptor aggregation by the use of hapten. Fig. 3 shows the effects of these manipulations on Ag-stimulated changes in [Ca2+]i. Fig. 3A illustrates the effect on [Ca2+]i after the addition of EGTA. At doses leading to similar initial increases in [Ca2+]i, the difference in the later persistence of the elevation is apparent. The addition of EGTA only slightly decreased the initial rise, but eliminated the subsequent persistent elevation; LaCl3 (Fig. 3B) gave similar results. The dependence of the persistent elevation of [Ca2+]i on aggregation was confirmed by disaggregating the receptors with hapten (Fig. 3C).

Role of [Ca2+]i in transcription of the MCP-1 gene and subsequent events

Methodological aspects. We first investigated whether we could substitute the simple method that measures MCP-1 protein for the more cumbersome Northern blot assay for mRNA. This would allow us to measure more samples and potentially post-transcriptional events as well. Our previous studies showed that increasing doses of Ag added to the RBL-2H3 cells sensitized with specific IgE stimulated the accumulation of mRNA for MCP-1 (Fig. 4, inset). Monitoring the accumulation of MCP-1 protein, albeit in cell suspensions rather than adherent cells as in the earlier study, showed a similar proportionality over a similar range of doses for both the slowly dissociating (high affinity) and the rapidly dissociating (low affinity) ligand (Fig. 4) That is, at lower doses the rapidly dissociating ligand stimulated a disproportionately lower synthesis of MCP-1 protein per unit phosphorylated Fc{epsilon}RI, but at higher doses it appeared to be as effective as the more slowly dissociating ligand.



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  		FIGURE 4. Total production of MCP-1 in cells stimulated with varying doses of the alternative Ags. Cells were incubated with anti-DNP IgE and then stimulated for 4 h with either the DNP- or NP-modified Ag. Cells were lysed by adding Triton X-100 (0.03%) to the samples, and the total MCP-1 protein was measured by ELISA assay. Upper abscissa, DNP Ag ({square}); lower abscissa, NP Ag ({triangleup}). The mean values (±SD) from three independent experiments performed in duplicate are shown. The absolute concentrations of MCP-1 detected in samples stimulated with 10 ng/ml DNP (corresponding to the value of 1 in the figure) were 2.6, 1.9, and 1.3 ng/106 cells, respectively. Inset, MCP-1 mRNA stimulated by variable doses of the low and high affinity ligands (data from Ref.3 ). Symbols are the same as in the main figure.

 
In the earlier study we also showed that adding actinomycin D 1 h after stimulating the cells promptly abrogated further accumulation of MCP-1 mRNA, but that this level was maintained for at least 1 h (3). In the current work we examined total production of MCP-1 protein in stimulated cells before and after the addition of actinomycin D (Fig. 5). The resting RBL-2H3 cells contained little of the chemokine (5, 6), and in the absence of stimulation there was little or no increase over the course of the experiment ({circ}). In the stimulated cells the production of MCP-1 increased throughout the observation period (•). This was also true of the cells stimulated for 60 min and then reacted with actinomycin ({blacksquare}), but the total MCP-1 produced by the latter cells became progressively less compared with that produced by cells in which transcription was allowed to continue. This is the expected result if the stimulation induces progressive transcription of mRNA, and the mRNA is relatively stable and is translated into protein at a constant rate throughout the experiment. Adding actinomycin prevented further accumulation of mRNA, but not the continued translation of the existing mRNA (see Discussion). Together these results indicate that MCP-1 protein can serve as a surrogate for monitoring the mRNA for the chemokine. Furthermore, in the presence of actinomycin, assaying MCP-1 protein in the entire cell suspension reflects translation of the mRNA produced at the time transcription was halted, whereas by assaying only the supernatant we can independently monitor the secretion of the protein.



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  		FIGURE 5. Kinetics of accumulation of MCP-1 protein. Cells were stimulated with 50 ng/ml DNP Ag. After 1 h, 2 µg/ml actinomycin D was added to some samples ({blacksquare}), but not others (•), and the total MCP-1 protein was measured in the lysed samples taken at the times indicated. {circ}, Samples treated with neither Ag nor actinomycin. The results are representative of two equivalent experiments. Error bars show the range of values.

 
Transcription of the MCP-1 gene. We were readily able to confirm previous reports that raising intracellular Ca2+ by inhibiting the Ca2+-ATPase responsible for reuptake of Ca2+ into endoplasmic reticulum (13) stimulates transcription of MCP-1 (5). To examine further the role of Ca2+ in receptor-initiated transcription, we tested whether the transcription required an influx of extracellular Ca2+. We stimulated cells with 10 ng/ml DNP Ag in the presence of medium containing 1.8 mM Ca2+, with or without other additions (Fig. 6). After 75 min, specimens were taken, and the remaining cells were washed, resuspended in medium with or without other additions, and resampled after an additional 45, 75, and 105 min.



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  		FIGURE 6. Effect of extracellular Ca2+ on the production of MCP-1. Cells were stimulated with 10 ng/ml DNP Ag in the presence ({blacksquare} and {square}) or the absence ({blacktriangleup} and {triangleup}) of external Ca2+. After 75 min, 1.8 mM Ca2+ was added to those samples in Ca2+-free medium, and transcription was terminated by adding 2 µg/ml actinomycin D in some ({blacksquare} and {blacktriangleup}). The MCP-1 protein that accumulated during the subsequent 105 min was measured. • and {circ}, Unstimulated cells incubated in Ca2+-containing medium. Stimulation of the cells in Ca2+-free medium during the entire length of the experiment did not yield MCP-1 levels above the background values (not shown). Depletion of free Ca2+ in the medium was achieved by chelating it with 3 mM EGTA (A) or using Ca2+-free medium (B). A second experiment gave very similar results, except that the effects of depletion of Ca2+ and addition of EGTA were indistinguishable.

 
As already noted, cells not exposed to Ag or to any additives (Fig. 6A, {circ}) produced little or no MCP-1, those stimulated with Ag throughout but not to any additives ({square}) produced substantial chemokine, and those stimulated with Ag throughout and with actinomycin beginning at 75 min produced less, but still a substantial amount of MCP-1 ({blacksquare}). The difference in MCP-1 produced by the cells treated or not with actinomycin reflects the additional mRNA available for translation during the second incubation. Another aliquot of cells was exposed to 3 mM EGTA during the initial 75 min. (This concentration of EGTA was sufficient to prevent any Ag-stimulated release of {beta}-hexosaminidase from cells in separate experiments (data not shown).) Thereafter, the cells were washed free of the chelator and exposed to Ag in Ca2+-containing medium ({triangleup}). These cells had synthesized no MCP-1 by 75 min, but produced progressive amounts thereafter. The latter amounts were similar to the difference in production observed by the cells treated or not with actinomycin ({square} and {blacksquare}; see above). This result indicates that EGTA reversibly prevented the stimulated transcription of the gene for MCP-1. That EGTA prevented transcription is demonstrated by the failure of those cells exposed to actinomycin during the second period (Fig. 6A, {blacktriangleup}) to generate any MCP-1. That is, if the EGTA had failed to block transcription, then during the second incubation the cells in the protocol shown by {blacktriangleup} should have synthesized MCP-1 roughly comparably to those subjected to the protocol shown by {triangleup}. Similar results were obtained using a Ca2+-free medium (Fig. 6B) rather than adding EGTA to the usual medium to expose the cells to decreased free Ca2+. In the results of the experiment illustrated in Fig. 6B by {triangleup}, it appears that depletion of Ca2+ inhibited transcription less than addition of EGTA. This was not a reproducible finding in additional experiments. Additional control experiments demonstrated that the responses observed after Ca2+ was replenished required the addition of Ag and were not mimicked by simply restoring Ca2+ (data not shown).

To test whether such a depletion of [Ca2+]i rather than a lack of influx of extracellular Ca2+ was the primary effect of these perturbants, we used the channel blocker, LaCl3, at a concentration (100 µM) that reduced exocytosis by 90 ± 5.6% (n = 4) compared with control samples. The lanthanide was added 1 min before stimulation with Ag, and the total accumulation of MCP-1 was assessed after 2 h of incubation with Ag. As documented in the left bars in Fig. 7, LaCl3 failed to inhibit biosynthesis of the chemokine, indicating normal transcription of the gene for MCP-1. These results suggest that the inhibition of transcription noted in the experiments with EGTA or Ca2+-deficient medium largely resulted from depletion of intracellular Ca2+, rather than simply from preventing an influx of Ca2+ from extracellular sources.



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  		FIGURE 7. Effect of LaCl3 on Ag-induced MCP-1 response. Anti-DNP-sensitized cells were preincubated for 1 min with 100 µM LaCl3 before stimulation with 50 ng/ml DNP Ag. After 2 h of incubation, total production and secretion of MCP-1 protein were measured in the lysate of the cell suspension and that of the cell-free supernatants, respectively. The bars show the mean ± SD from three independent experiments performed with duplicate samples.

 
To determine whether we could directly observe the differential effect of EGTA, and LaCl3 on intracellular Ca2+, we performed the following experiment. Cells loaded with Fura Red and fluo-4 were exposed to buffer control, 3 mM EGTA, or 100 µM LaCl3 and stimulated with Ag. After 75-min incubation at room temperature, the fluorescence of both dyes was measured by FACS analysis (Fig. 8). The fluorescence of the cells stimulated in the presence of LaCl3 was very similar to that of cells stimulated in the absence of additives, indicating that these cells retained similar levels of [Ca2+]i. However, the cells exposed to EGTA had a markedly lower fluorescence compared with the control or the sample treated with LaCl3, indicating lower levels of [Ca2+]i. To each sample we added 4 mM CaCl2 (77 min after addition of Ag). Notably, only the cells that had been exposed to EGTA showed a marked increase in fluorescence intensity. Thus, the addition of Ca2+ in sufficient excess to overcome EGTA restored [Ca2+]i to the level seen in the control cells and those treated with LaCl3.



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  		FIGURE 8. Effects of EGTA and LaCl3 on poststimulation levels of [Ca2+]i. Cells were pretreated with 3 mM EGTA, 100 µM LaCl3, or nothing shortly before the addition of DNP Ag. Fluorescence was monitored beginning 75 min after the addition of Ag, and at 77 min Ca2+ was added to each of the samples to a final concentration of 4 mM.

 
We also tested the effect of disaggregating the receptor under conditions of stimulation with Ag and at a time during the response when the [Ca2+]i cannot be distinguished from that seen with unstimulated cells (Fig. 3C). Cells were stimulated with 50 ng/ml DNP Ag, and the effects of adding hapten after 1 h were compared with the effects of adding 2 µg/ml actinomycin or nothing. Consistent with our previous results using adherent cells (3), the addition of hapten was as effective as actinomycin in halting further transcription when added 1 h after the addition of Ag (data not shown). In control experiments the same dose of hapten had no such effect on transcription stimulated by CPA.

Translation of MCP-1 mRNA. In protocols similar to those described in Figs. 6 and 7, we added EGTA as well as actinomycin after 1 h. There was little inhibition of MCP-1 production for the first 20 min, but thereafter the inhibition was definite, although variable ({triangleup} in Fig. 9A). The results shown by the left columns in Fig. 7 indicate that blocking influx of Ca2+ with lanthanide failed to inhibit either transcription or translation, suggesting that translation, like transcription, requires little or no influx of extracellular Ca2+. In this instance also, the effect of EGTA is likely to have resulted from its depletion of intracellular Ca2+.



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  		FIGURE 9. Effect of EGTA on translation and secretion of MCP-1. A, Effect on translation. Cells were stimulated for 1 h with 50 ng/ml DNP Ag before 2 µg/ml actinomycin D ({square}) or actinomycin D plus 3 mM EGTA ({triangleup}) were added to the samples. The total production of MCP-1 was measured during the following hour of incubation. Mean values (±SD) for six independent experiments performed in duplicate are shown. B, Effect on secretion. Cells were treated as described in A. After stimulation with the DNP Ag, the cells were exposed to actinomycin D ({square}) or actinomycin D plus EGTA ({triangleup}), and the incubation was continued for another hour. During the second part of stimulation the amount of MCP-1 was determined in the cell-free supernatants. The values are the average of duplicate samples from four experiments. The error bars show the SDs.

 
Secretion of MCP-1. We also examined the requirements for the secretion of MCP-1 by separately assaying MCP-1 in medium and cells. As shown in Fig. 9B, the addition of EGTA strongly inhibited the secretion of MCP-1. In four experiments the mean decrease was 72 ± 4.7% (±SEM). Again, this appears largely due to depletion of intracellular Ca2+, because simply blocking entry of Ca2+ with LaCl3 had virtually no effect (right bars, Fig. 7). The inhibition of secretion was not statistically greater than the inhibition of total production.

Roles of PLC{gamma} and PKC. Considerable experimental evidence supports the current model in which receptor-stimulated phosphorylation of Syk kinase results successively in the activation of phospholipase C{gamma} (PLC{gamma}), the production of inositol 1,4,5-trisphosphate (IP3), and the release of Ca2+ from IP3-sensitive stores (14). The molecular mechanisms that lead to the subsequent opening and regulation of channels that allow for influx of Ca2+ from the extracellular milieu are less well defined and may involve more than one pathway (15).

If the responses to the manipulations we used were indeed related to changes in [Ca2+]i and not to some other effect of the perturbants we employed, we would expect that an inhibitor of PLC{gamma} would likewise suppress the expression of MCP-1 protein. We incubated IgE-loaded cells with U-73122 (16) or its inactive structural analog, U-73343, after 5 min stimulated them with Ag for 2 h, and then measured the total MCP-1 and the amount released. At a concentration of 10 µM, the dose used for short term measurements (17), both the active and the inactive control compound sharply suppressed production of MCP-1 (not shown). However, by reducing the doses a clear distinction between the inhibitor and its inactive analog was observed (Fig. 10). In the three experiments the mean inhibition at the 1-µM dose was 64 ± 11.7% (±SEM); the inhibition of secretion was 88 ± 9.6%. Pooling all data from five experiments for various doses, the effect on secretion was greater than that on production of the chemokine (p < 0.01).



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  		FIGURE 10. MCP-1 response in stimulated RBL-2H3 cells in the presence of an inhibitor of PLC{gamma}. Cells were incubated with the indicated concentrations of U-73122, U-73343, or buffer for 5 min and exposed to DNP Ag (50 ng/ml). Supernatants and lysates of cell suspensions were analyzed after 2 h of stimulation. Total accumulation ({blacksquare}) and secretion ({square}) of MCP-1 relative to the values in the control, Ag-stimulated cells. The absolute values for MCP-1 were 0.69 and 0.35 ng/106 cells for production and secretion in these samples, respectively. A single representative of three experiments is shown. The error bars show the range of duplicate specimens.

 
To test further whether the action of inhibitors of PLC{gamma} was simply due to their inhibition of the PLC{gamma}-dependent increase in [Ca2+]i, we treated cells with ATPase inhibitors, which release Ca2+ from internal stores not by generating IP3 but by poisoning the uptake mechanism. As expected, the inhibitors of PLC{gamma} affected the increase in [Ca2+]i by Ag but little or not at all the increase stimulated by the inhibitors, DTBQ and CPA. Nonetheless, the MCP-1 synthesis stimulated by the latter agents was inhibited substantially (data not shown).

Activated PLC{gamma} and, in particular, phospholipase D generate diacylglycerol, which along with Ca2+ activates protein kinase C (PKC) (18). It was therefore of interest to determine whether the MCP-1 response was also affected by the activation of PKC. Cells were incubated with the staurosporin analog Ro-31-8220, an inhibitor of PKC (19) (although known to have additional actions other than on PKC (20)), or with its inactive analog, bisindolylmaleimide V, for 30 min and then exposed to Ag over 2 h. Fig. 11A shows that the inhibitor, but not its inactive analog, inhibited MCP-1 production in a dose-dependent manner. A related analog, Ro-31-8425, that is a more specific inhibitor of PKC (21, 22) gave comparable results (not shown). Therefore, the production of MCP-1 does appear to be PKC dependent. Further probing indicated a more complex picture, however. When MCP-1 synthesis was stimulated by two Ca2+-ATPase inhibitors that raise intracellular Ca2+ by promoting its release from internal stores, the MCP-1 response was similarly completely inhibited by the PKC inhibitor (Fig. 11B). Control experiments (not shown) indicated that the PKC inhibitor had little or no effect on the increase in [Ca2+]i stimulated either by Ag or the ATPase inhibitors.



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  		FIGURE 11. MCP-1 response in the presence of an inhibitor of PKC. A, Cells were preincubated for 30 min with or without increasing micromolar concentrations of Ro-31-8220 or bisindolylmaleimide V before stimulation with DNP Ag (50 ng/ml). Accumulated MCP-1 was determined in the lysates of cell suspensions collected after 2 h. {square}, MCP-1 produced by cells exposed only to the additives; {blacksquare}, cells stimulated with Ag in the presence of additives. B, Cells were exposed for 30 min to 3 µM Ro-31-8220 or 3 µM bisindolylmaleimide V before stimulation with DNP Ag (50 ng/ml), DTBHQ (10 µM), or CPA (3 µM). MCP-1 in the samples was determined after 2 h. The accumulation of MCP-1 in the presence of Ro-31-8220 or bisindolylmaleimide V is shown relative to the levels in cells stimulated with DNP ({blacksquare}), DTBHQ ({square}), or CPA (), respectively, in the absence of additives. No increase in MCP-1 above the baseline level was detected in samples exposed to Ro-31-8220 before incubation with the triggering agents. The absolute amounts of MCP-1 in the samples stimulated with DNP, DTBHQ, and CPA were 1.1, 0.25, and 1.0 ng/106 cells, respectively. The results shown are from one experiment, with the error bars showing the range of duplicate specimens. A second experiment gave equivalent results.

 
Influence of receptor aggregates

In our previous work we observed that monovalent hapten relatively promptly aborted synthesis of MCP-1 mRNA stimulated by the addition of a polyvalent hapten conjugate to RBL cells sensitized with IgE (3). In the current work we monitored total cellular MCP-1 protein instead of mRNA in a similar protocol. Remarkably, there was little difference between the amount of MCP-1 protein produced by the cells after they had been reacted with either actinomycin or hapten (Fig. 12, {blacksquare} and {blacktriangleup}). This result indicates that whereas transcription of the gene for MCP-1 requires persistent aggregation of the Fc{epsilon}RI, translation of the transcript does not. On the other hand, MCP-1 secretion was largely inhibited by disaggregating the receptors (65 ± 3.2% at 2 h; {triangleup}).



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  		FIGURE 12. Effect of hapten on the Ag-induced MCP-1 response. Cells were stimulated for 1 h with 50 ng/ml DNP Ag before 2 µg/ml actinomycin D ({square} and {blacksquare}) or actinomycin D plus 100 µM hapten ({blacktriangleup} and {triangleup}) were added to the samples. During the following hour of stimulation MCP-1 was determined in the lysates of cell suspension ({blacksquare} and {blacktriangleup}) and of the cell-free supernatants ({square} and {circ}). {circ}, Secreted MCP-1 from unstimulated cells. The data are from a single experiment and show the means and range of duplicate samples. Three additional experiments gave equivalent results.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The purpose of these studies was to explore further the question of how a late response to activation of Fc{epsilon}RI could be relatively little constrained by kinetic proofreading, whereas some earlier responses clearly were sensitive to this regulatory mechanism. Specifically, our earlier work had demonstrated that the efficiency with which a given number of phosphorylated receptors could stimulate the relatively proximal phosphorylation of Syk kinase was markedly influenced by the binding characteristics of the ligand that induced the aggregation by binding to the receptor-bound IgE (2); later steps, such as degranulation, were even more markedly sensitive to the binding properties of the ligand. On the other hand, transcription of the gene for MCP-1, which persisted even hours after the cells were stimulated, showed a lesser sensitivity and then only at lower dosages (3).

We proposed that a branch point in the cascade initiated by the receptor might account for this phenomenon. Such a branch might generate a soluble messenger and even if that messenger was itself short-lived, then, provided the more weakly binding (rapidly dissociating) ligand could stimulate a sufficient level of the messenger, the system would have the characteristics observed experimentally. A formal model for such a mechanism demonstrated the conditions under which such a mechanism would quantitatively account for the observed effects (4).

On the basis of earlier work on stimulation of MCP-1 by others (5, 6), we decided to examine the role of Ca2+ as a, or the, candidate messenger. Our results strengthen the likelihood that Ca2+ play such a role.

We found that even the rapidly dissociating NP conjugate could generate a substantial increase in [Ca2+]i. The pattern of the change in [Ca2+]i in the cell population stimulated by the NP conjugate at first appeared to be distinctive. That is, whereas the more slowly dissociating DNP Ag showed an initial peak, followed by a more or less prolonged persistent smaller elevation, doses of the NP Ag yielding a similarly sized initial elevation showed a lesser persistent phase. However, with both ligands these patterns were highly sensitive to the dose used. That is, at sufficiently low doses of either ligand, the initial rise was both smaller and delayed, whereas the persistent elevation actually seemed to be higher and more prolonged than at higher doses. This probably simply reflects that at the lower doses we are seeing the composite effect of sequential signaling of cells, each of which undergoes a rise and fall in [Ca2+]i, whereas at the higher doses there is a virtually simultaneous signaling of all cells. Experiments at the single-cell level would be required to determine whether instead this phenomenon reflected a differential activation of a negative feedback pathway (15).

Stimulation of Fc{epsilon}RI is known to open store-operated channels for Ca2+ (23). Two manipulations that should block such an influx of Ca2+, adding EGTA to the medium or depleting the medium of Ca2+, largely inhibited the transcription of MCP-1. However, lanthanide ions at concentrations sufficient to block the secretion of hexosaminidase failed to effectively block transcription. These results can be harmonized if the Ca2+-free or EGTA-containing medium served to deplete [Ca2+]i below a critical level, a level that appears not to require much influx of Ca2+ from the outside.

If the results of the perturbations we used were due to their effect on Ca2+ rather than on some unanticipated target, then the relatively specific inhibitor of PLC{gamma}, the enzyme thought to be critically important for stimulating the rise in Ca2+ by Fc{epsilon}RI, should likewise inhibit the formation of MCP-1. The observed inhibition of the receptor-stimulated rise in [Ca2+]i and the simultaneous inhibition of MCP-1 biosynthesis were consistent with the critical role of Ca2+ in receptor-mediated stimulation of MCP-1 biosynthesis. However, the fact that the inhibitors of PLC{gamma} were equivalently effective in inhibiting the production of MCP-1 stimulated by compounds that do not depend on that lipase to generate the rise in [Ca2+]i shows that the inhibitors are acting at multiple sites. Likewise, inhibitors of PKC (which, unlike the inhibitors of PLC{gamma}, did not inhibit the rise of [Ca2+]i stimulated by aggregating the receptors or the inhibitors of ATPase) inhibited the synthesis of MCP-1 promoted by both stimuli. The first result is consistent with a simple model, but inhibition of the response to the ATPase inhibitors likewise complicates the interpretation of the effect.

There are experimental data showing that the activation of MCP-1 transcription principally involves NF-{kappa}B (24, 25), and Dolmetsch and Lewis (26) have documented that, at least in T lymphocytes, activation of NF-{kappa}B is related more to the rapid transient rises than to the sustained increases in [Ca2+]i. This would be consistent with the ability of the more rapidly dissociating ligand to stimulate MCP-1 production at doses that give a good transient rise in [Ca2+]i.

We cannot exclude the possibility that pathways distal to the Ca2+ shunt must also be activated (or are already active) and that the Ca2+ acts principally to create permissive conditions for the products of such pathways. However, this does not negate the importance of the concept that systems constrained by kinetic proofreading can generate messengers that permit certain cellular responses to escape the constraint.

The indirect method we used to assess transcription of the MCP-1 gene allowed us simultaneously to assess the subsequent translation of the message and the secretion of the chemokine. The striking finding was that whereas maneuvers that diminished [Ca2+]i inhibited the transcription, translation, and secretion of MCP-1, none of these processes was substantially inhibited by blocking the influx of Ca2+ using doses of lanthanide sufficient to virtually completely block receptor-mediated secretion of hexosaminidase (Table I). These results suggest a substantially lower requirement for [Ca2+]i for each of the major steps leading to secretion of the chemokine.


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  		Table I. Effect of perturbants on Fc{varepsilon}RI-stimulated biosynthesis of MCP-1

 
A surprising finding was that whereas transcription and secretion of MCP-1 depend on signals generated by aggregated Fc{epsilon}RI, the intervening step, translation of the preformed mRNA for MCP-1, continues relatively normally even when the receptors are disaggregated. Although still sensitive to depletion of Ca2+ stores, the level of [Ca2+]i required for this process must be very low. With respect to the sensitivity of the transcription and secretion of MCP-1 to the state of aggregation of the receptors, the mechanism could involve more than simply interfering with Ca2+. As shown in Fig. 3, the levels of Ca2+ in cells stimulated at the higher doses of Ag especially (Fig. 3C) drop fairly rapidly to baseline values. Nevertheless, continued transcription continues to be sensitive to the depletion of Ca2+ even after >1 h (Fig. 6). Although it is likely that the inhibition observed upon addition of hapten relates to the receptor-induced Ca2+ signal, we cannot rule out that other necessary signals may likewise require continued aggregation of the receptors. Exactly the same considerations relate to the secretion of MCP-1.

Together our experimental findings are consistent with the mechanism we proposed for escape from the kinetic proofreading, and underscore the prediction (4) that quantitative differences in the strength of the signal generated by ligands of differing affinity can lead to qualitatively distinctive cellular responses.

Evidence for an alternative escape from the constraints of kinetic proofreading has been presented by Rosette et al. (27), who studied early and late responses of T cells. Surprisingly, a low affinity, rapidly dissociating ligand stimulated late responses even though it stimulated early responses much less effectively than a tighter binding ligand. They proposed a "trickling through" mechanism (similar to that anticipated by McKeithan (1)) in which a later, largely irreversible, step gradually becomes occupied, in effect acting as a counter. Such a mechanism does not seem to account for the ability of the rapidly dissociating ligand to stimulate transcription of the gene for MCP-1, since that remains highly sensitive to disaggregation of the Fc{epsilon}RI (3).


   Acknowledgments
 
We thank Chikako Torigoe for the experiments illustrated in Fig. 1, and Michael Beaven for a careful reading of the manuscript and useful suggestions.


   Footnotes
 
1 Current address: 1450 Caballero, San Antonio, TX 78224. Back

2 Address correspondence and reprint requests to Dr. Henry Metzger, Room 9N-228, 10 Center Drive, National Institute of Arthritis and Musculoskeletal Diseases, National Institutes of Health, Bethesda, MD 20892-1820. E-mail address: metgerh{at}exchange.nih.gov Back

3 Abbreviations used in this paper: MCP-1, monocyte chemotactic protein-1; [Ca2+]i, intracellular Ca2+ concentration; CPA, cyclopiazonic acid; DAG, diacylglycerol; DTBHQ, 2,5-di-tert-butylhydroquinone; IP3, inositol-1,4,5-trisphosphate; NP, 2-nitrophenyl; PLC{gamma}, phospholipase C{gamma}; PKC protein kinase C. Back

Received for publication April 22, 2002. Accepted for publication December 18, 2002.


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